Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method

Cecilia Bozzetti, Federico Quaini, Anna Squadrilli, Marcello Tiseo, Caterina Frati, Costanza Lagrasta, Cinzia Azzoni, Lorena Bottarelli, Maricla Galetti, Angela Alama, Silvana Belletti, Rita Gatti, Antonio Passaro, Angela Gradilone, Andrea Cavazzoni, Roberta Alfieri, Pier Giorgio Petronini, Mara Bonelli, Angela Falco, Cecilia CarubbiGiuseppe Pedrazzi, Rita Nizzoli, Nadia Naldi, Carmine Pinto, Andrea Ardizzoni

Research output: Contribution to journalArticle

Abstract

Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

Original languageEnglish
Article numbere0142891
JournalPLoS One
Volume10
Issue number11
DOIs
Publication statusPublished - Nov 1 2015

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Circulating Neoplastic Cells
squamous cell carcinoma
Cell Adhesion Molecules
Squamous Cell Neoplasms
cell adhesion
Tumors
Squamous Cell Carcinoma
lungs
Cells
Lung Neoplasms
Receptor, Fibroblast Growth Factor, Type 1
Lung
lung neoplasms
Epithelial-Mesenchymal Transition
cells
Tissue Donors
methodology
Blood
Leukocytes
epithelial cells

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Bozzetti, C., Quaini, F., Squadrilli, A., Tiseo, M., Frati, C., Lagrasta, C., ... Ardizzoni, A. (2015). Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method. PLoS One, 10(11), [e0142891]. https://doi.org/10.1371/journal.pone.0142891

Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method. / Bozzetti, Cecilia; Quaini, Federico; Squadrilli, Anna; Tiseo, Marcello; Frati, Caterina; Lagrasta, Costanza; Azzoni, Cinzia; Bottarelli, Lorena; Galetti, Maricla; Alama, Angela; Belletti, Silvana; Gatti, Rita; Passaro, Antonio; Gradilone, Angela; Cavazzoni, Andrea; Alfieri, Roberta; Petronini, Pier Giorgio; Bonelli, Mara; Falco, Angela; Carubbi, Cecilia; Pedrazzi, Giuseppe; Nizzoli, Rita; Naldi, Nadia; Pinto, Carmine; Ardizzoni, Andrea.

In: PLoS One, Vol. 10, No. 11, e0142891, 01.11.2015.

Research output: Contribution to journalArticle

Bozzetti, C, Quaini, F, Squadrilli, A, Tiseo, M, Frati, C, Lagrasta, C, Azzoni, C, Bottarelli, L, Galetti, M, Alama, A, Belletti, S, Gatti, R, Passaro, A, Gradilone, A, Cavazzoni, A, Alfieri, R, Petronini, PG, Bonelli, M, Falco, A, Carubbi, C, Pedrazzi, G, Nizzoli, R, Naldi, N, Pinto, C & Ardizzoni, A 2015, 'Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method', PLoS One, vol. 10, no. 11, e0142891. https://doi.org/10.1371/journal.pone.0142891
Bozzetti, Cecilia ; Quaini, Federico ; Squadrilli, Anna ; Tiseo, Marcello ; Frati, Caterina ; Lagrasta, Costanza ; Azzoni, Cinzia ; Bottarelli, Lorena ; Galetti, Maricla ; Alama, Angela ; Belletti, Silvana ; Gatti, Rita ; Passaro, Antonio ; Gradilone, Angela ; Cavazzoni, Andrea ; Alfieri, Roberta ; Petronini, Pier Giorgio ; Bonelli, Mara ; Falco, Angela ; Carubbi, Cecilia ; Pedrazzi, Giuseppe ; Nizzoli, Rita ; Naldi, Nadia ; Pinto, Carmine ; Ardizzoni, Andrea. / Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method. In: PLoS One. 2015 ; Vol. 10, No. 11.
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abstract = "Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89{\%}) SQCLC patients. Thirty-four of the 44 (77{\%}) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43{\%} (18/42) of pooled circulating SQCLC cells, whereas only 29{\%} (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.",
author = "Cecilia Bozzetti and Federico Quaini and Anna Squadrilli and Marcello Tiseo and Caterina Frati and Costanza Lagrasta and Cinzia Azzoni and Lorena Bottarelli and Maricla Galetti and Angela Alama and Silvana Belletti and Rita Gatti and Antonio Passaro and Angela Gradilone and Andrea Cavazzoni and Roberta Alfieri and Petronini, {Pier Giorgio} and Mara Bonelli and Angela Falco and Cecilia Carubbi and Giuseppe Pedrazzi and Rita Nizzoli and Nadia Naldi and Carmine Pinto and Andrea Ardizzoni",
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T1 - Isolation and characterization of circulating tumor cells in squamous cell carcinoma of the lung using a non-EpCAM-based capture method

AU - Bozzetti, Cecilia

AU - Quaini, Federico

AU - Squadrilli, Anna

AU - Tiseo, Marcello

AU - Frati, Caterina

AU - Lagrasta, Costanza

AU - Azzoni, Cinzia

AU - Bottarelli, Lorena

AU - Galetti, Maricla

AU - Alama, Angela

AU - Belletti, Silvana

AU - Gatti, Rita

AU - Passaro, Antonio

AU - Gradilone, Angela

AU - Cavazzoni, Andrea

AU - Alfieri, Roberta

AU - Petronini, Pier Giorgio

AU - Bonelli, Mara

AU - Falco, Angela

AU - Carubbi, Cecilia

AU - Pedrazzi, Giuseppe

AU - Nizzoli, Rita

AU - Naldi, Nadia

AU - Pinto, Carmine

AU - Ardizzoni, Andrea

PY - 2015/11/1

Y1 - 2015/11/1

N2 - Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

AB - Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

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